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. Author manuscript; available in PMC: 2015 Sep 29.
Published in final edited form as: Dev Cell. 2014 Sep 4;30(6):645–659. doi: 10.1016/j.devcel.2014.07.001

Local CRH signaling promotes synaptogenesis and circuit integration of adult-born neurons

Isabella Garcia a,b, Kathleen B Quast c, Longwen Huang d, Alexander M Herman a, Jennifer Selever c, Jan M Deussing f, Nicholas J Justice g, Benjamin R Arenkiel a,c,d,h,*
PMCID: PMC4182170  NIHMSID: NIHMS626301  PMID: 25199688

Summary

Neural activity either enhances or impairs de novo synaptogenesis and circuit integration of neurons, but how this activity is mechanistically relayed in the adult brain is largely unknown. Neuropeptide-expressing interneurons are widespread throughout the brain and are key candidates for conveying neural activity downstream via neuromodulatory pathways that are distinct from classical neurotransmission. With the goal of identifying signaling mechanisms that underlie neuronal circuit integration in the adult brain, we have virally traced local Corticotropin-releasing hormone (CRH)-expressing inhibitory interneurons with extensive presynaptic inputs onto new neurons that are continuously integrated into the adult rodent olfactory bulb. Local CRH signaling onto adult-born neurons promotes and/or stabilizes chemical synapses in the olfactory bulb, revealing a neuromodulatory mechanism for continued circuit plasticity, synapse formation, and integration of new neurons in the adult brain.

Keywords: neuropeptides, CRH, CRF, olfactory, granule cell, synapse, circuit, activity, plasticity, GPCR, EPL

Introduction

Synaptogenesis and circuit integration of neurons in the brain is governed by diverse repertoires of synaptic and extrasynaptic inputs. Excitatory input by principal neurons has profound effects on sculpting and pruning synaptic connectivity (Favero and Castro-Alamancos, 2013; Le Roux et al., 2013). However, recent evidence suggests that interneurons also play significant roles in modulating synapse formation (De Marco Garcia et al., 2011; Hensch et al., 1998; Le Magueresse and Monyer, 2013). Inhibitory interneurons are highly heterogeneous, and depending on the brain region, can vastly outnumber principal neurons (Chen and Greer, 2004; Isaacson and Strowbridge, 1998; Lledo et al., 2008). Neurochemical classification schemes have shown that interneurons not only express GABA and calcium-binding proteins such as Parvalbumin (PV), Calretinin (CR), and Calbindin (Barinka et al., 2012; Kosaka and Kosaka, 2008; Rudy et al., 2011), but also a cast of neuromodulatory peptides, including Somatostatin (SST), Cholecystokinin, Oxytocin, and Corticotropin-releasing hormone (CRH) (Huang et al., 2013; Le Magueresse and Monyer, 2013; Ma et al., 2006; Rudy et al., 2011; Xu et al., 2013). Neuropeptidergic interneurons are promising candidates for modulating changes in local synapse and circuit structure, and are pervasive throughout the rodent olfactory bulb (OB).

Endowed with the feature of ongoing neurogenesis (Alvarez-Buylla and Temple, 1998), the mouse olfactory system offers an excellent in vivo model to investigate mechanisms that underlie synaptogenesis, circuit plasticity, and the integration of new neurons into existing networks (Abrous et al., 2005; Ming and Song, 2005). Adult-born neurons are continuously generated in the subventricular zone (SVZ), migrate via the rostral migratory stream (RMS), and populate the OB where the vast majority become inhibitory granule cells that form connections with OB principal mitral and tufted cells (Mori et al., 1983; Price and Powell, 1970b; Carleton et al., 2003; Shepherd and Greer, 2004). This interaction influences olfactory behaviors and odor-related memories (Abraham et al., 2010; Breton-Provencher et al., 2009; Mouret et al., 2009; Rochefort et al., 2002).

Studies have found that the survival and integration of adult-born granule cells is activity-dependent during a developmental critical period between two and four weeks after their birth (Kelsch et al., 2009; Yamaguchi and Mori, 2005), when they receive inputs from local interneuron subtypes, including deep and superficial short axon cells and Blanes cells (Arenkiel et al., 2011; Eyre et al., 2008; Pressler and Strowbridge, 2006), as well as centrifugal fibers from deeper regions of the brain (Arenkiel et al., 2011; Balu et al., 2007; Panzanelli et al., 2009; Whitman and Greer, 2007). Maturing granule cells extend their dendrites into the external plexiform layer (EPL), where they connect with principal mitral cells. Interestingly, the EPL also harbors a more dispersed, and heterogeneous population of neuropeptidergic interneurons (Kosaka and Kosaka, 2008; Lepousez et al., 2010a,b) that also form reciprocal synaptic connectivity with mitral cells (Huang et al., 2013; Kato et al., 2013; Miyamichi et al., 2013). Unlike granule cells, EPL interneurons are generated in the early postnatal period and remain stable throughout life (Batista-Brito et al., 2008), but their potential neuromodulatory role in shaping the integration of adult-born neurons is unknown.

We have previously shown that odor enrichment increases the number of inputs onto adult-born neurons in the OB (Arenkiel et al., 2011), causing enhanced cell survival and integration. However, the precise signaling mechanisms between these inputs and granule cells remain in question. In this study, we have mapped local neuropeptidergic EPL interneurons with anatomical and functional connectivity onto granule cells during peak periods of synaptogenesis and circuit integration. Using loss- and gain-of-function analyses, conditional viral-genetic technologies, optical imaging, electrophysiological recordings, and optogenetic stimulation, we have uncovered a neuropeptidergic signaling mechanism between local CRH+ EPL interneurons and adult-born granule cells that plays an important role in synapse formation, circuit plasticity, and the integration of new neurons into the existing networks, revealing a dual functional role for neuropeptidergic inhibitory interneurons in the mouse OB.

Results

Adult-born neurons receive inputs from local EPL interneurons

To reveal the identities of the presynaptic inputs made onto adult-born granule cells, we performed targeted monosynaptic tracing using genetically engineered Rabies Virus (RV), SADG-EGFP RV (Arenkiel et al., 2011; Wickersham et al., 2007a,b). RV is a neurotropic virus that travels retrogradely between connected neurons. Endowing it with the avian coat protein EnvA provides selectivity of infection to ‘source’ cells that express the TVA receptor, and replacing the glycoprotein G with an EGFP reporter in the viral genome provides a fluorescently labeled map of connectivity. Using a conditional knock-in mouse (ROSALSL-Rabies G-IRES-TVA, referred to herein as ROSARITVA/RITVA) that expresses the elements for conditional monosynaptic tracing (rabies-G and TVA) (Takatoh et al., 2013), we selectively targeted adult-born neurons in the OB for transsynaptic tracing. ROSARITVA/RITVA mice were stereotaxically injected with a lentivirus that expressed Cre recombinase and a tdTomato reporter into the RMS (Figure 1A). TdTomato marked adult-born neurons red, and Cre recombinase allowed for conditional expression of TVA and G. 28 days post lentiviral injection, we introduced RV into the core of the OB to target newly integrated granule cells for connectivity mapping. One week post RV infection, the targeted OB showed high level EGFP expression (Figure 1B) compared to negative controls in which no Cre was present (Figure S1A), suggesting that 1) adult-born neurons were successfully targeted for tracing, and 2) that retrograde jumping of RV had occurred. Tissue sections revealed strong expression of tdTomato in adult-born ‘source’ cells, and high efficiency RV labeling in presynaptic inputs (Figure 1C-D). As positive controls, we identified presynaptic EGFP labeling in mitral cells (Figure 1D), validating RV jumping to known synaptic partners. Interestingly, we found that adult-born granule cells also received extensive inputs from both deep and superficially located interneurons, with highest enrichment from cells located in the EPL (Figure 1D).

Figure 1. Adult-born granule cells receive presynaptic inputs from external plexiform layer interneurons.

Figure 1

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(A) Genetic strategy for targeting adult-born neurons for transsynaptic tracing. (B) Dorsal view of mouse brain showing a labeled OB 7 days post infection with SADG-EGFP RV (scale bar 500 μm). (C) Cross-section of (B) (RMS-rostral migratory stream, GCL-granule cell layer, EPL-external plexiform layer, GL-glomerular layer; scale bar 300 μm). (D) High magnification view of (C). tdTomato+/EGFP+ cells (arrows) are newborn neuron ‘source cells’. EGFP+ cells are presynaptic inputs (open arrowheads mark EPL presynaptic inputs, asterisks mark mitral cell inputs; MCL-mitral cell layer; scale bar 80 μm). Inset shows a ‘source cell’ (scale bar 15 μm). (E) EPL presynaptic inputs are PV+ (arrows, scale bar 10 μm), and CRH+ (F) (arrows, arrowheads mark extracellular CRH, scale bar 10 μm). See also Figure S1.

Local CRH+ EPL interneurons make connections onto adult-born granule cells

Immunohistochemical characterization of the EGFP+ EPL cells revealed that granule cell inputs constituted a subset of non-dopaminergic interneurons (GFAP-0%, Tyrosine Hydroxylase-0%, CR+ 92% ± 5% (Figure S1B-D), with strong co-labeling of PV (95% ± 3%, Figure 1E), and SST (45% ± 5%, Figure S1E). Highest neuropeptide expression was observed with CRH (68% ± 4%, Figure 1F), suggesting that adult-born neurons received direct input from resident CRH+ interneurons in the OB. Although immunohistochemistry identified enrichment of CRH protein in EPL interneurons, we also detected substantial extracellular CRH in the EPL (Figure 1F), suggesting that CRH is locally secreted. To more precisely identify CRH+ interneurons as bona fide inputs, we employed genetic labeling techniques.

To this end, we crossed CRH-Cre mice to conditional tdTomato reporter mice (CRH-Cre+/−; ROSALSL-tdTom+/-), and observed strong tdTomato signal in the paraventricular nucleus of the hypothalamus, a known hub for CRH synthesis and secretion (Figure S2), and high levels of expression in EPL interneurons of the OB (Figure 2A), which we previously characterized as multipolar and anaxonic fast-spiking PV+ interneurons (Huang et al., 2013). To determine if adult-born granule cells received inputs from CRH+ EPL interneurons, we performed transsynaptic tracing in CRH-Cre+/−; ROSALSL-tdTom+/− mice and electroporated the avian TVA receptor and rabies-G into the SVZ of CRH-Cre+/−; ROSALSL-tdTom+/− pups (Figure 2B), transiently targeting neural progenitors that give rise to adult-born neurons. 28 days later, mice were injected with RV into the core of the OB, and sacrificed 7 days later. OB slices revealed strong EGFP expression, with high efficiency infection of targeted granule ‘source cells’ and presynaptic inputs (Figure 2C). Both ‘source cells’ and all inputs were labeled EGFP+, whereas CRH+ inputs were EGFP+/tdTomato+. Through this differential labeling, we verified that new granule cells indeed received extensive input from local CRH+ EPL interneurons with 86% (± 5% SEM) of EPL presynaptic inputs expressing CRH via lineage tracing.

Figure 2. Local CRH+ EPL interneurons make connections onto adult-born granule cells.

Figure 2

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(A) Genetic lineage of CRH+ neurons in the OB of CRH-Cre+/−; ROSALSL-tdTom+/− mice (GL-glomerular layer, EPL-external plexiform layer, MCL-mitral cell layer, GCL-granule cell layer, scale bars 500, 150, and 25 μm). (B) Experimental scheme to identify presynaptic inputs in CRH lineage traced mice. (C) SADΔG-EGFP RV transsynaptic tracing in CRH-Cre+/−; ROSALSL-tdTom+/− mice (arrows identify newborn neuron ‘source cells’, open arrowheads mark CRH+ presynaptic EPL interneurons, scale bars 150 and 80 μm). See also Figure S2.

Adult-born granule cells dynamically express CRHR1

CRH is best known as a hypothalamic regulatory hormone that mediates systemic stress responses (Vale et al., 1981; Vale et al., 1983). In addition, CRH has been implicated both as a neurotransmitter and neuromodulator in the hippocampus, amygdala, and cerebellum (Maras and Baram, 2012; Roozendaal et al., 2008; Schmolesky et al., 2007). CRH can bind to two G-protein coupled receptors, CRHR1 and CRHR2 (Perrin et al., 1993; Perrin et al., 1995), but in the brain it has been shown to interact with higher affinity to CRHR1, which mediates many of its physiological effects (Bale and Vale, 2004). When bound to CRHR1, Gs-coupled signaling is activated (Berger et al., 2006; Blank et al., 2003; Perrin et al., 1993; Thiel and Cibelli, 1999).

Having identified that CRH+ EPL interneurons provide inputs onto granule cells, we next sought to investigate the expression of CRH receptors in the OB. RT-PCR revealed that both CRH and CRHR mRNAs were present in the bulb, and that CRHR1 was expressed at much higher levels than CRHR2 (Figure 3A). Because available antibodies for CRHR are not useful for detecting endogenous CRHR1 (Refojo et al., 2011), and that the expression pattern is not conclusive in the OB (Figure S3A), we utilized genetic strategies to determine the CRHR1 cell type-specific expression pattern. Consistent with mRNA transcript detection, tissue sections from CRHR1-EGFP BAC transgenic mice, whose expression pattern was previously validated to match endogenous CRHR1 (Justice et al., 2008), revealed high levels of spatially restricted CRHR1 in granule cells (Figure 3B). Finally, to determine the precise spatial localization of CRH and CRHR1 neurons within the OB, CRHR1-EGFP mice were crossed to CRH-Cre+/−; ROSALSL-tdTom+/− mice to generate CRHR1-EGFP+/−; CRH-Cre+/−; ROSALSL-tdTom+/− double reporter mice. OB sections showed a cell type-specific juxtaposition between EGFP-labeled CRHR1+ granule cells, and tdTomato-labeled CRH+ EPL interneurons (Figure 3C). Whereas CRHR1+ granule cell bodies were located throughout the GCL with superficial enrichment, CRH+ EPL interneurons almost exclusively resided in the EPL, were absent from the GCL (Figure S3B), and directly juxtaposed CRHR1+ dendrites. These data support the idea that CRH ligand is locally released by EPL interneurons and directly acts on CRHR1+ granule cell dendrites in the EPL.

Figure 3. Adult-born granule cells dynamically express CRHR1.

Figure 3

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(A) Semi-quantitative RT-PCR for CRH and CRHR1/2 of whole OB RNA. (B) OB cross-section of CRHR1-EGFP BAC transgenic mice (RMS-rostral migratory stream, GCL-granule cell layer, EPL-external plexiform layer, GL-glomerular layer, scale bar 200 μm). (C) Reporter expression of CRHR1-EGFP; CRH-Cre+/−; ROSALSL-tdTom+/− mice (arrows point to CRHR1+ granule cells, open arrowheads mark CRH+ EPL interneurons, scale bars 60 and 20 μm). (D) Experimental scheme to determine the developmental expression profile of CRHR1 expression in granule cells. (E) CRHR1-expression in newborn neurons 28 days post EdU injection (scale bar 60 μm). (F) Quantification of CRHR1-expression in granule cells (data points represent averages +/− SEM, n=3 animals per time point). (G) CRHR1::EGFP expression in adult-born granule cells (scale bars 100 and 20 μm). See also Figure S3.

Interestingly, we noted that EGFP expression was nearly absent from the RMS and became gradually enriched in the outer GCL, with strong enrichment in superficial regions (Figure 3B-C). These data suggested that CRHR1 might exhibit a dynamic expression pattern with granule cell maturation, and that CRHR1 might be expressed both in early postnatal-born granule cells which predominantly localize to the superficial GCL (Lemasson et al., 2005), as well as adult-born granule cells. To test if CRHR1 is dynamically regulated during periods of newborn granule cell synaptogenesis, and better determine its spatiotemporal expression profile, CRHR1-EGFP mice were pulsed with the proliferation marker EdU, and sacrificed at time points between 7-60 days, spanning early, intermediate, and late phases of synaptogenesis and circuit integration (Figure 3D) (Carleton et al., 2003). We found that the ratio of EdU-labeled granule cells that expressed CRHR1 was very low at 7 days of age (5.8 % ± 3.9 %, Figure 3E-F), and substantially increased between 14 (34.5 % ± 4.1 %), 21 (59.2 % ± 4.2 %), and 28 days of age (81.3 % ± 2.2 %). This value slightly increased further at 40 days post-EdU pulsing (87.8 % ± 4.5 %), and the number of co-labeled neurons plateaued at 60 days (87.5 % ± 3.2 %). Intriguingly, dynamic enrichment of CRHR1 coincided with critical periods of activity-dependent survival, synaptogenesis, and circuit function between 14 and 28 days of granule cell age (Kelsch et al., 2009; Mouret et al., 2008; Yamaguchi and Mori, 2005).

Finally, to determine the subcellular localization of CRHR1, we expressed a CRHR1::EGFP fusion construct with a tdTomato cell fill in new granule cells. We observed CRHR1::EGFP in dendrites, with enriched subcellular localization in a subset of dendritic spines in the EPL (Figure 3G). Interestingly, CRHR1::EGFP was also present in extra-synaptic dendritic regions (Figure S3C-E), suggesting that CRH-mediated local neuropeptide signaling might not occur exclusively at synapses, but also via extra-synaptic mechanisms not restricted to dendritic spine structures.

Together, these data support the idea that CRH is synthesized locally by EPL interneurons and can signal to granule cells via time-dependent expression of CRHR1, suggesting a possible role for secreted CRH in the long-term survival and circuit integration of adult-born neurons.

CRH signaling is required for normal levels of adult-born granule cell survival

To determine how CRH signaling affects adult-born neurons, mice lacking CRH or its receptor (CRH−/− or CRHR1−/− ) were pulsed with BrdU and sacrificed either 24 hours later to assay proliferation in the SVZ (BrdU+/Ki67+ cells), or 30 days later to assay survival in the GCL (BrdU+ cells, Figure 4A), focusing on deeper regions where adult-born granule cells reside (Lemasson et al., 2005). Compared to wildtype littermates, CRH−/− mutants showed increased SVZ proliferation (p<0.05, 4942 ± 302 cells in CRH−/−, compared to 3906 ± 354 cells in CRH+/+ mice), but decreased cell survival in the OB (p<0.005, 25 ± 1 cell in CRH−/−, compared to 36 ± 2 cells in CRH+/+ mice) (Figure 4B and C). This increased proliferation in CRH−/− mice is consistent with previous reports that stress impairs neurogenesis both in the SVZ and in the hippocampus (de Andrade et al., 2013; Hitoshi et al., 2007, Schoenfeld and Gould, 2013), and that CRH−/− mice show decreased stress levels (Jacobson et al., 2000). Cleaved caspase-3 and TUNEL staining revealed increased apoptosis in the GCL of CRH−/− mice (Figure S4A). To investigate whether granule cell apoptosis was secondary to loss of CRH+ interneurons, we examined the integrity of the EPL and performed cell counts using DAPI and CR, which overlaps with EPL interneurons (Huang et al., 2013, Figure S1D and S4B-C) and found no difference. Moreover, because CRH has important systemic effects as a regulatory hormone, many of which are mediated by corticosteroids, we questioned whether the attrition of granule cells in CRH−/− mice was corticosteroid-dependent and supplemented adult CRH−/− mice with corticosterone. Corticosteroid supplementation at a concentration that readily crosses the blood brain barrier and is capable of rescuing embryonic phenotypes in utero (Muglia et al., 1995), did not change granule cell survival in CRH−/− mice (Figure S4F), suggesting that adult-born neuron survival was not mediated by systemic CRH signaling, but likely through local CRH.

Figure 4. CRH signaling is required for normal levels of adult-born granule cell survival and synaptic protein expression.

Figure 4

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(A) Experimental scheme to determine cellular proliferation and granule cell survival in CRH mutant alleles (scale bars 100 and 15 μm). (B) Quantification of proliferating cells (BrdU and Ki67 double-positive cells) in the SVZ of control and CRH−/− mice (* p< 0.05 Student’s t-test). (C) Quantification of adult-born granule cell survival in control and CRH−/− mice. (D) Quantification of proliferating cells in the SVZ of CRHR1−/− mice (p>0.05 Student’s t-test). (E) Quantification of adult-born granule cell survival in control and CRHR1−/− mice (*p<0.001 Student’s t-test). (F) Representative images of the GCL of CRHR1+/+ and CRHR1f/f mice that expressed Cre-EGFP or tdTomato in granule cells (scale bar 50 μm). (G) Quantification of the ratio of Cre-EGFP+/tdTom+ granule cells (* p<0.01 Student’s t-test). (H-P) Western blots of the synaptic proteins Synapsin, PSD95, and NR2B of OBs of CRHR1−/−, CRHR1−/−, and CRHR1f/f mice injected with Cre or control viruses (* p<0.05 Student’s t-test). All data points averages +/− SEM, n=4 animals each. See also Figure S4.

As loss of secreted CRH systemically in CRH−/− mice could have secondary effects in the OB, we next assayed for proliferation and cell survival in CRHR1−/− mice, and saw no change in SVZ-based proliferation compared to control littermates (p>0.05, 4172 ± 113 cells in CRHR−/− vs. 4337 ± 303 cells in CRHR+/+ mice, Figure 4D). These data were consistent with the observation that the SVZ lacked CRHR1 expression in CRHR1-EGFP mice (data not shown), and that SVZ proliferation might be mediated by systemic signaling rather than through central CRH receptor activation. However, decreased numbers of granule cells were noted in the OB 30 days post BrdU pulsing (p<0.001, 24 ± 1 cells in CRHR−/− vs. 32 ± 2 cells in CRHR+/+ mice, Figure 4E), as well as increased numbers of apoptotic cells (Figure S4D). Moreover, this phenotype was also corticosteroid-independent (Figure S4G).

In order to bypass any potential systemic effects of using germline CRH loss-of-function alleles, we next conditionally removed CRHR1 specifically from adult-born granule cells by injecting a mixture of equal titers of AAV particles that expressed either Cre-P2A-EGFP or tdTomato (control) into the RMS of CRHR1+/+ or CRHR1flox/flox mice (Kuhne et al., 2012), and revealed a 24.8 ± 2.8 % decrease in the ratio of Cre-EGFP+/tdTom+ granule cells between CRHR1+/+ and CRHR1flox/flox mice (Figure 4F-G). Interestingly, morphological analysis on the proportion of surviving granule cells showed no difference between Cre and tdTomato+ neurons (data not shown).

Having found decreased numbers of surviving granule cells in CRH loss-of-function mutants, we wondered if synaptic protein expression was affected in these models and performed Western blot analysis, which showed significantly decreased levels of Synapsin, PSD95, and NR2B in the OBs of CRH−/−, CRHR−/−, and CRHR1flox/flox mice compared to controls (Figure 4H-P). Together, these data imply an important role for CRH signaling in the maintenance and/or generation of synapses in the OB.

We next asked if CRHR1 expression correlates with cell survival, and stained CRHR1-EGFP olfactory bulb tissue with activated caspase-3 (Figure S4H). Interestingly, we did not observe any CRHR1-EGFP+/caspase-3+ cells. Together, these loss-of-function and molecular marker data suggest that granule cells require local CRH signaling for normal development and maturation, and that CRHR1 expression correlates with synapse formation and/or survival, allowing them to integrate into existing brain circuits.

Gain-of-function CRH signaling promotes increased synaptic protein expression in the OB

Having found that lack of CRH signaling leads to impaired granule cell survival (Figure 4), we next queried the consequence of enhanced CRH signaling. To determine a role for CRHR1 in shaping synapse development and neuronal maturation, we expressed a constitutively active version of CRHR1 fused to EGFP ((CA)CRHR::EGFP) (Nielsen et al., 2000) in new granule cells (Figure 5A). Morphological characterization of granule cells expressing (CA)CRHR::EGFP revealed normal average neuron length comparable to tdTomato controls (p>0.05, 284 ± 21 μm in (CA)CRHR1:EGFP vs. 316 ± 19 μm in controls, Figure 5B), but increased total dendrite length (p<0.05, 894 ± 30 μm vs. 672 ± 25 μm, Figure 5C), dendritic branch number (p<0.01, 10.5 ± 0.65 μm vs. 7 ± 0.7 μm, Figure 5D), and branch intersections at both proximal and distal radii from the soma (p<0.05, Figure 5E). Next, we quantified dendritic spines and found that total spine number was increased in (CA)CRHR1:EGFP neurons (Figure 5F). Neurolucida reconstructions revealed increased complexity in (CA)CRHR1::EGFP granule cells compared to control neurons (Figure 5G).

Figure 5. Constitutive CRHR-signaling in granule cells promotes synaptogenic changes in the OB.

Figure 5

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(A) Representative images of granule cells expressing tdTomato or tdTomato and a constitutively-active CRHR1-EGFP fusion construct ((CA)CRHR::EGFP, scale bar 50 μm). (B) Quantification of average granule cell length (p>0.05 Student’s t-test), (C) mean total dendrite length (* p<0.05), and (D) average dendritic branch number in tdTomato and (CA)CRHR+ granule cells (*p<0.01 Student’s t-test). (E) Scholl analysis of the number of intersections in tdTomato or (CA)CRHR+ granule cells (*p<0.05 ANOVA). (F) Quantification of the number of dendritic spines between tdTomato and (CA)CRHR+ granule cells (*p<0.05 ANOVA). N=10 cells each from 3 animals. (G) Representative granule cell morphology reconstructions. (H) Experimental scheme for targeting CRHR1+ OB granule cells for constitutive CRHR1-activation. (I) Expression pattern of AAV-flex-(CA)CRHR::GFP in granule cells (arrows point to (CA)CRHR::GFP+ neurons, scale bars 1000, 100, and 20 μm). (J-O) Western blot analysis of synaptic protein expression of CRHR-Cre+/− OBs injected with either flexed GFP or flexed-(CA)CRHR-GFP AAV (* p<0.05 Student’s t-test, n=4 animals each). All data points averages +/− SEM. See also Figure S5.

Then, we targeted granule cells in the OB for conditional gain-of-function studies with spatiotemporal specificity during periods of endogenous CRHR1 expression to determine if CRHR1 activation is sufficient for modulating synaptogenic changes. For this, we first generated a BAC transgenic allele to drive Cre recombinase from the CRHR1 promoter. To validate cell type specificity of Cre expression, we generated CRHR1-EGFP;CRHR1-Cre+/;ROSALSL-tdTom+/− mice, and saw Cre activity that matched the expression pattern of CRHR1-EGFP transgenic mice in the OB (Figure S5A). We next generated a conditional adeno-associated virus that carries (CA)CRHR1::EGFP in an inverted “flexed” configuration (Atasoy et al., 2008), and stereotaxically targeted the RMS for infection of granule cells in CRHR1-Cre+/− mice (Figure 5H-I). To confirm enhanced signaling through CRHR1, we isolated whole OBs for Western blot analysis. Control lysate was obtained from CRHR1-Cre+/− mice injected with serotype-matched flexed GFP virus. Since CRHR1 is Gs-coupled and leads to activation of CREB via phosphorylation (Blank et al., 2003; Thiel and Cibelli, 1999), we first assayed pCREB levels in (CA)CRHR1-expressing olfactory bulbs (Figure 5J), and observed significantly increased levels of pCREB with (CA)CRHR1 expression. Interestingly, we also observed significant changes in levels of synaptic protein expression, including a large increase in the presynaptic protein Synapsin (Figure 5K), suggesting the formation of de novo synapses or the strengthening of existing synapses with enhanced CRHR1 signaling. Intriguingly, however, although the quantity of PSD95 showed an increased trend of expression, these changes were not statistically significant (Figure 5L). Because granule cells are GABAergic and form reciprocal dendrodendritic synapses with mitral cells (Mori et al., 1983; Panzanelli et al., 2009; Shepherd and Greer, 2004), we also assayed for changes in both inhibitory and excitatory receptor expression. The mitral cell-specific GABA-Aα1 (Whitman and Greer, 2007), as well as NR1, and NR2B receptor subunits showed increased expression (Figure 5M-N and S5B), whereas levels of AMPA receptor subtypes were decreased compared to GFP controls (Figure 5O and S5C). Immunohistochemistry for the upregulated proteins revealed increased levels of NMDA and GABA receptor expression in the EPL of gain-of-function experiments compared to controls (Figure S5D-F). Intriguingly, increased NR1 and NR2B expression was localized to both (CA)CRHR1::EGFP+ as well as EGFP lacking dendritic structures, which could be due to the nature of the membrane-bound overexpression construct. For example, the fusion protein is not exclusively targeted to all synaptic structures within newborn granule cells, and its synaptogenic properties may in fact influence the formation of synaptic structures in a more widespread manner. Consistent with this, we also note significant upregulation of GABA-Aα1, which is expressed by mitral/tufted cells in the OB, and is not expected to colocalize with (CA)CRHR::EGFP structures. Together, these data suggest an overall increase in synaptic connectivity within the OB circuitry with constitutive CRHR1 signaling, which then also may lead to non cell-autonomous increases in synaptic protein expression.

Together, increased spine numbers, upregulated Synapsin and NMDA receptor expression, combined with decreased AMPA receptors suggests that CRH signaling in the OB promotes the formation of new immature synapses, and/or potential synaptic scaling of existing excitatory synapses (Turrigiano et al., 1998; Turrigiano and Nelson, 2004).

Gain-of-function CRH signaling promotes functional synaptogenesis in the OB

A hallmark of bona fide circuit integration is functional synaptic connectivity. Having determined that constitutively active CRHR1 signaling leads to synaptogenic changes in the OB (Figure 5), we next queried the functional consequence of gain-of-function CRH signaling using electrophysiology.

To determine if soluble CRH ligand influenced granule cell electrophysiological properties, we made whole cell recordings from CRHR1-EGFP expressing granule cells while bath-applying CRH, which showed no change in the frequency of miniature excitatory postsynaptic currents (mEPSCs), but induced a significant decrease in amplitude (p<0.05, 11.55 ± 0.72 pA before, and 8.8 ± 0.35 pA after CRH, Figure 6A-D). Whole cell recordings from granule cells that expressed (CA)CRHR1 compared to EGFP controls showed no change in mEPSC frequency, but significantly decreased mEPSC amplitudes (p<0.05, 7.73 ± 1.38 pA in (CA)CRHR1 vs.11.69 ± 1.27 pA in EGFP controls, Figure 6E-H). These data were consistent with the observation that CRH gain-of-function signaling through activated CRHR1 led to decreased AMPA receptor levels (Figure 5O and S5C) and further suggested that upregulation of glutamatergic synapses via NMDA receptor expression likely reflects functionally silent or immature synapses. Worth noting, we did not observe any rapid changes in firing, passive membrane properties, or membrane potential following CRH application (data not shown), suggesting that in the OB CRH does not act directly as a neurotransmitter, but likely functions as a true neuromodulator.

Figure 6. Constitutive CRHR-signaling in granule cells promotes synaptic and circuit plasticity in the OB.

Figure 6

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(A) Representative trace of CRHR1-EGFP+ granule cell before and after CRH application (500 nM CRH). (B) Average mEPSCs before and after CRH. (C-D) Quantification of the mEPSC frequency and amplitude before and after CRH bath application (*p<0.05, n = 13 cells from 3 animals). (E) Representative mEPSC traces of granule cells from CRHR-Cre+/− mice injected with either AAV-flexed EGFP or AAV-flexed-(CA)CRHR::EGFP. (F) Average mEPSCs of EGFP and (CA)CRHR1::EGFP+ granule cells. (G-H) Quantified granule cell mEPSC frequency and amplitude (*p<0.05 Student’s t-test, n = 11 granule cells per group from 3 animals). (I) Representative mIPSC traces of mitral cells from CRHR-Cre+/− mice injected with either AAV-flexed EGFP or AAV-flexed-(CA)CRHR::EGFP. (J) Average mitral cell mIPSCs. (K-L) Average frequency and amplitude of mitral cell mIPSCs in CRHR-Cre+/− OBs in which CRHR-expressing granule cells express either EGFP or (CA)CRHR1::EGFP (* p<0.05 Student’s t-test, n = 13 mitral cells per group from 3 animals). All data points represent mean ±SEM.

With the observation that GABA-Aα1 receptor subunit expression increased with CRH signaling (Figure 5M), we next hypothesized that functional GABAergic synapses from granule cells onto mitral cells might be upregulated. To address this, we recorded miniature inhibitory postsynaptic currents (mIPSC) from mitral cells in OBs where CRHR+ granule cells expressed (CA)CRHR::EGFP, and found a significant increase in frequency of mIPSCs (p<0.05, 2.21 ± 0.39 Hz in (CA)CRHR1 vs. 1.17 ± 0.18 pA in EGFP controls, Figure 6I-L), suggesting either increased formation or stabilization of granule cell-mitral cell synapses or changes in presynaptic release properties. Hence, biochemical and electrophysiological evidence suggests that enhanced CRHR signaling in granule cells leads to increased functional synaptogenesis and circuit plasticity in the OB.

Acute optogenetic activation of CRH+ EPL interneurons promotes release of CRH in the OB

CRH+ EPL interneurons make connections onto granule cells, which in concert dynamically express CRHR1 (Figures 1-3). Lack of CRHR1 expression during a critical time window in granule cell maturation and circuit integration leads to decreased cell survival (Figure 4), and constitutively active CRHR1 enhances synapse formation and circuit integration of adult-born neurons (Figures 5 and 6). Together these data support a mechanism by which activity-induced release of CRH from EPL interneurons may influence granule cell synaptogenesis. We next questioned if manipulating the activity of CRH+ EPL interneurons directly and acutely could dynamically recapitulate the physiological effects observed with constitutive CRHR1 activation.

To activate CRH+ EPL interneurons with spatiotemporal specificity, CRH-Cre+/− mice were crossed to ROSALSL-ChR2 mice to obtain CRH-Cre+/−; ROSALSL-ChR2 mice that selectively expressed the light gated cation channel channelrhodopsin 2 (ChR2) (Boyden et al., 2005; Nagel et al., 2003) in CRH+ EPL interneurons in the OB. From these mice, we made slices of the OB (Figure 7A) and hypothalamus (Figure S6), which were acutely photostimulated ex vivo in small volumes of artificial cerebral spinal fluid (ACSF). Light-stimulated depolarization of CRH+ neurons led to CRH release in both hypothalamic control (Figure 7B) and OB slices (Figure 7C), whereas soluble CRH levels in light-stimulated controls were unchanged. Together, these data suggest that the targeted depolarization of CRH+ neurons elicited the release of stored CRH neuropeptide from EPL interneurons.

Figure 7. Optogenetic activation of CRH+ EPL interneurons induces synaptogenesis in the OB.

Figure 7

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(A) ChR2 expression pattern of CRH-Cre+/−; ROSALSL-ChR2+/− mice (scale bars 200 and 50 μm). (B-C) Quantification of CRH concentration with hypothalamic or OB optogenetic activation in ROSALSL-ChR2+/− (control) or CRH-Cre+/−; ROSALSL-ChR2+/− mice (*p<0.05 Student’s t-test, n = 3 animals per group). (D) Experimental scheme for in vivo photostimulation of CRH+ EPL interneurons. (E-H) Western blot analysis of synaptic protein expression of ROSALSL-ChR2+/− (control) or CRH-Cre+/−; ROSALSL-ChR2+/− mice (*p<0.05 Student’s t-test, n = 4 animals per group). (I) Model: CRH signaling between local EPL interneurons and granule cells promotes synapse formation and stabilization in the OB. All data points represent mean ±SEM. See also Figure S6.

Finally, to determine the effects of CRH+ EPL interneuron activation on granule cell synapses in vivo, CRH-Cre+/−; ROSALSL-ChR2+/− mice were chronically implanted with fiber optics directly over the olfactory bulb (Ung and Arenkiel, 2012) and photostimulated with blue laser light (Figure 7D). Acute in vivo photostimulation in awake and behaving mice led to enhanced olfactory bulb expression of pCREB (Figure 7E), recapitulating the effect seen in constitutive CRHR1 activation (Figure 5J). Western blots of stimulated animals showed significant upregulation of the synaptic proteins Synapsin, PSD95, and NR2B (Figure 7F-H), suggesting a conserved synaptogenic effect of photostimulated release of CRH from EPL interneurons in vivo, which mirrored constitutively active CRHR1 signaling (Figure 5).

Discussion

Neuropeptides, including CRH, have been implicated in a variety of neuronal processes, ranging from neuromodulation and dendritic outgrowth, to neuroprotection (Chen et al., 2004; Hanstein et al., 2008; Sheng et al., 2012). However, the precise role of CRH in the maturation and integration of granule cells in the OB has not been investigated. Here, we describe a neuropeptidergic function for inhibitory interneurons in shaping cell survival, synaptogenesis, and circuit integration of new neurons in the adult brain that is distinct from classical neurotransmitter signaling. Together, our data support a potential mechanism for a tripartite-like interaction between mitral cells, EPL interneurons, and granule cells (Figure 7I). We and others have recently shown that mitral cells and EPL interneurons exhibit reciprocal connectivity (Huang et al., 2013, Kato et al., 2013; Miyamichi et al., 2013). It is conceivable that neuronal activity via olfactory sensation is conveyed to mitral cells, which in turn stimulate EPL interneurons to both shape mitral cell output, and release soluble CRH. In this way, activity-dependent release of CRH, and subsequent reception by granule cells that express CRHR1 both synaptically and/or extra-synaptically, may function as a key mechanism towards modulating synaptogenesis and circuit integration via neuropeptide signaling, linking activity to synaptogenesis and circuit integration via neuropeptidergic interneurons. Ultimately, this interaction has the potential to shape whole circuit activity and plasticity. Although this model supports a role for local interactions, it remains to be determined if other forms of neuromodulatory signals are conveyed specifically to EPL interneurons via other local or centrifugal inputs.

Dual roles of interneurons

Neuropeptides are traditionally acknowledged for roles in shaping whole-body physiological responses and/or modulating systemic homeostatic mechanisms (Vale et al., 1981). Recently, neuropeptides have received increasing attention for their roles in shaping synapses and facilitating neuronal plasticity (Bayatti et al., 2003; Fenoglio et al., 2006; Lipschitz et al., 2005). Interestingly, however, interneurons have been most extensively studied with respect to their traditional, GABAergic inhibitory function, and the neuropeptides they express have served primarily in categorizing the many different interneuron subtypes throughout the brain (Ma et al., 2006; Rudy et al., 2011; Xu et al., 2013). Although granule cells make up the majority of inhibitory interneurons in the OB, previous work from our lab and others has identified other types of interneurons with GABAergic connections onto mitral cells that likely serve important olfactory functions (Huang et al., 2013; Kato et al., 2013; Miyamichi et al., 2013; Lepousez et al., 2010ab; Kosaka and Kosaka, 2008). Here, we have identified a population of inhibitory interneurons in the OB with a neuropeptidergic role in promoting synaptic protein expression and circuit integration. Interestingly, this interneuron population has a dual role in shaping the OB circuitry and likely, olfaction. This interaction is inhibitory onto mitral cells and neuromodulatory onto granule cells, suggesting increased connectivity and/or signaling not only between excitatory principal cells and inhibitory interneurons, but also between different subtypes of interneurons. This apparent dual functionality may indeed exemplify a prominent mechanism to support neuronal plasticity, and a way to shape circuitries by inhibitory neurotransmitter signaling onto mitral cells, and neuromodulatory signaling onto granule cells. EPL interneurons form reciprocal GABAergic connections with mitral cells, and are in turn depolarized following odor stimulation (Huang et al., Kato et al., 2013; Miyamichi et al., 2013). This activation further strengthens mitral cell inhibition, but may also facilitate CRH release with depolarization, linking olfactory-dependent activity to neuromodulation, and ultimately synaptogenesis and circuit integration.

Local neuropeptide signaling, synaptogenesis, and circuit integration

Our data suggest that local neuromodulatory signaling by CRH+ EPL interneurons in the OB aids in granule cell circuit integration by increasing synaptic protein expression and/or stabilizing existing synapses. Removing CRH signaling decreases synaptic protein expression, whereas enhancing CRH signaling induces synaptic protein expression in the OB. Perhaps more intriguingly, the result that CRHR loss-of-function does not completely abolish adult-born neuron survival and integration poses some additional questions. Given that numerous neuropeptides, including Somatostatin, Vasointestinal peptide, Cholecystokinin, Oxytocin, and neuropeptide Y are present in the olfactory bulb (Gracia-Llanes et al., 2003; Lepousez et al., 2010ab; Ma et al., 2013; Tobin et al., 2010), and that granule cells in turn appear to express the cognate receptors, it remains to be seen if other neuromodulators have the same effect on the OB circuitry, and if these neuropeptides act in concert to promote synaptogenesis and circuit integration in the adult brain.

Experimental Procedures Experimental animals

All experimental animals were treated in compliance with the United States Department of Health and Human Services and the Baylor College of Medicine IUACUC guidelines. ROSARITVA/RITVA (Takatoh et al., 2013), ROSALSL-tdTom (Arenkiel et al., 2011), CRHR1-EGFP mice (Justice et al., 2008), and CRHR1flox/flox mice (Kuhne et al., 2012) were previously described. CRH-Cre (Taniguchi et al., 2011), ROSALSL-ChR2 (Madisen et al., 2012), CRH−/− (Muglia et al., 1995), and CRHR1−/− (Smith et al., 1998) were obtained from Jackson Laboratories and maintained on a C57BL/6 background. Generation of the CRHR1-Cre line is described in Supplemental Experimental Procedures.

Transsynaptic Tracing

Briefly, adult (6-8 week old) ROSARITVA/RITVA mice were injected with high titer lentivirus encoding tdTomato-IRES-Cre stereotaxically into the RMS. 28 days later, mice were injected with low titer SADG-EGFP RV (Wickersham et al., 2007b) into the core of the OB and sacrificed after 7 days for mapping studies. Next, CRH-Cre+/−; ROSALSL-tdTom+/− pups were electroporated with an expression construct for rabies-G and TVA as previously described (Arenkiel et al., 2011). 28 days post-electroporation, mice were injected with SADG-EGFP RV as described above. See also supplemental materials.

Immunohistochemistry and Imaging

OB tissues were processed for immunohistochemistry as previously described (Huang et al., 2013). Briefly, free-floating sections were stained with the following primary antibodies:, guinea pig anti-Parvalbumin, and rabbit anti-CRH (kindly provided by Nick Justice), followed by washing and incubation with species-specific Alexa-633 secondary antibodies. Confocal image analysis was performed using a Leica TCS SPE confocal microscope. See also supplemental materials

CRH−/− and CRHR1−/− adult-born neuron proliferation, survival, and apoptosis

Age-matched adult (6-8 weeks old) male mice were injected with BrdU. For SVZ-based proliferation studies, animals were sacrificed 24 later as and 50 μm thick sections were taken throughout the SVZ and stained with mouse anti-BrdU and rabbit anti-Ki67 to assess proliferating cells. To assay granule cell survival, mice were sacrificed 30 days later and processed for immunohistochemistry using mouse anti-BrdU antibody. Images were taken in the middle and outer granule cell layer, avoiding the RMS and IPL/MCL regions, and total cell numbers were counted through serial sections. See also supplemental materials.

Constitutively active CRHR1 overexpression

A constitutively active CRHR1::EGFP fusion construct (Nielsen et al., 2000) was electroporated into the SVZ of P2 pups for morphology analysis using Neurolucida software. The construct was further subcloned into a conditional flexed adeno-associated-viral vector (Atasoy et al., 2008) and packaged into viral particles. Virus was stereotaxically injected into the RMS of CRHR1-Cre mice, and OB tissue was harvested 14 d post injection. See also supplemental materials.

Electrophysiology

Animals (P21-P35) were deeply anesthetized using isoflurane, and perfused intracardially with ice-cold artificial cerebrospinal fluid (ACSF). Coronal olfactory bulb slices (300 μm) were placed in a room-temperature chamber mounted on an Olympus upright microscope (BX50WI), and perfused with oxygenated ACSF. Cells were visualized using fluorescence and differential interference contrast imaging. Synaptic currents were recorded using cesium-methanesulfonate based internal solutions. See also supplemental materials.

In vivo photostimulation

Fiber optics were generated and implanted directly over the olfactory bulb as previously described (Ung and Arenkiel, 2012). Animals were allowed to recover from the surgery for 3 days prior to photostimulation. Photostimulation was performed for 3 hours using a blue laser source (CrystaLaser, Reno, NV) controlled by a Master-8 (A.M.P.I., Israel). See also supplemental materials.

Statistical Methods

Unless otherwise indicated, statistical comparisons between experimental groups were made using Student’s T-test and all error bars represent standard error of the mean (SEM).

Supplementary Material

1
2

Highlights.

  • Viral transsynaptic tracing reveals functional interneuron-neuron connectivity

  • Local CRH+ interneurons provide inputs to adult-born neurons

  • CRH signaling promotes adult-born neuron survival and circuit integration

  • Neuropeptidergic interneurons promotes synapse formation and function

Acknowledgements

This work was supported by the McNair Medical Institute, NINDS grant F31NS081805 to I.G. and NINDS R01NS078294 to B.R.A. We would like to thank Drs Dona Murphey, Ian Davison, and Steve Shea for critical review of this manuscript.

Footnotes

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References

  1. Abraham NM, Egger V, Shimshek DR, Renden R, Fukunaga I, Sprengel R, Seeburg PH, Klugmann M, Margrie TW, Schaefer AT, et al. Synaptic inhibition in the olfactory bulb accelerates odor discrimination in mice. Neuron. 2010;65:399–411. doi: 10.1016/j.neuron.2010.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: from precursors to network and physiology. Physiol Rev. 2005;85:523–569. doi: 10.1152/physrev.00055.2003. [DOI] [PubMed] [Google Scholar]
  3. Alvarez-Buylla A, Temple S. Stem cells in the developing and adult nervous system. J Neurobiol. 1998;36:105–110. [PubMed] [Google Scholar]
  4. Arenkiel BR, Hasegawa H, Yi JJ, Larsen RS, Wallace ML, Philpot BD, Wang F, Ehlers MD. Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing. PLoS One. 2011;6:e29423. doi: 10.1371/journal.pone.0029423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Atasoy D, Aponte Y, Su HH, Sternson SM. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J Neurosci. 2008;28:7025–7030. doi: 10.1523/JNEUROSCI.1954-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bale TL, Vale WW. CRF and CRF receptors: role in stress responsivity and other behaviors. Annu Rev Pharmacol Toxicol. 2004;44:525–557. doi: 10.1146/annurev.pharmtox.44.101802.121410. [DOI] [PubMed] [Google Scholar]
  7. Balu R, Pressler RT, Strowbridge BW. Multiple modes of synaptic excitation of olfactory bulb granule cells. J Neurosci. 2007;27:5621–5632. doi: 10.1523/JNEUROSCI.4630-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barinka F, Salaj M, Rybar J, Krajcovicova E, Kubova H, Druga R. Calretinin, parvalbumin and calbindin immunoreactive interneurons in perirhinal cortex and temporal area Te3V of the rat brain: qualitative and quantitative analyses. Brain Res. 2012;1436:68–80. doi: 10.1016/j.brainres.2011.12.014. [DOI] [PubMed] [Google Scholar]
  9. Batista-Brito R, Close J, Machold R, Fishell G. The distinct temporal origins of olfactory bulb interneuron subtypes. J Neurosci. 2008;28:3966–3975. doi: 10.1523/JNEUROSCI.5625-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bayatti N, Zschocke J, Behl C. Brain region-specific neuroprotective action and signaling of corticotropin-releasing hormone in primary neurons. Endocrinology. 2003;144:4051–4060. doi: 10.1210/en.2003-0168. [DOI] [PubMed] [Google Scholar]
  11. Berger H, Heinrich N, Wietfeld D, Bienert M, Beyermann M. Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different confromations of its J-domain. Br J Pharmacol. 2006;149:942–947. doi: 10.1038/sj.bjp.0706926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bicknell RJ, Leng G. Relative efficiency of neural firing patterns for vasopressin release in vitro. Neuroendocrinology. 1981;33:295–299. doi: 10.1159/000123248. [DOI] [PubMed] [Google Scholar]
  13. Blank T, Nijholt I, Grammatopoulos DK, Randeva HS, Hillhouse EW, Spiess J. Corticotropin-releasing factor receptors couple to multiple G-proteins to activate diverse intracellular signaling pathways in mouse hippocampus: role in neuronal excitability and associative learning. J Neurosci. 2003;23:700–707. doi: 10.1523/JNEUROSCI.23-02-00700.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci. 2005;8:1263–1268. doi: 10.1038/nn1525. [DOI] [PubMed] [Google Scholar]
  15. Breton-Provencher V, Lemasson M, Peralta MR, 3rd, Saghatelyan A. Interneurons produced in adulthood are required for the normal functioning of the olfactory bulb network and for the execution of selected olfactory behaviors. J Neurosci. 2009;29:15245–15257. doi: 10.1523/JNEUROSCI.3606-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Carleton A, Petreanu LT, Lansford R, Alvarez-Buylla A, Lledo PM. Becoming a new neuron in the adult olfactory bulb. Nat Neurosci. 2003;6:507–518. doi: 10.1038/nn1048. [DOI] [PubMed] [Google Scholar]
  17. Chen Y, Bender RA, Brunson KL, Pomper JK, Grigoriadis DE, Wurst W, Baram TZ. Modulation of dendritic differentiation by corticotropin-releasing factor in the developing hippocampus. Proc Natl Acad Sci U S A. 2004;101:15782–15787. doi: 10.1073/pnas.0403975101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Chen WR, Greer CA. Olfactory bulb. In: Shepherd GM, editor. The synaptic Organization of the Brain. 5th edition. Oxford University Press; New York: 2004. pp. 165–216. [Google Scholar]
  19. de Andrade JS, Cespedes IC, Abrao RO, Dos Santos TB, Diniz L, Britto LR, Spadari-Bratfisch RC, Ortolani D, Melo-Thomas L, da Silva RC, et al. Chronic unpredictable mild stress alters an anxiety-related defensive response, Fos immunoreactivity and hippocampal adult neurogenesis. Behav Brain Res. 2013;250:81–90. doi: 10.1016/j.bbr.2013.04.031. [DOI] [PubMed] [Google Scholar]
  20. De Marco Garcia NV, Karayannis T, Fishell G. Neuronal activity is required for the development of specific cortical interneuron subtypes. Nature. 2011;472:351–355. doi: 10.1038/nature09865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Eyre MD, Antal M, Nusser Z. Distinct deep short-axon cell subtypes of the main olfactory bulb provide novel intrabulbar and extrabulbar GABAergic connections. J Neurosci. 2008;28:8217–8229. doi: 10.1523/JNEUROSCI.2490-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Favero M, Castro-Alamancos MA. Synaptic cooperativity regulates persistent network activity in neocortex. J Neurosci. 2013;33:3151–3163. doi: 10.1523/JNEUROSCI.4424-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fenoglio KA, Chen Y, Baram TZ. Neuroplasticity of the hypothalamic-pituitary-adrenal axis early in life requires recurrent recruitment of stress-regulating brain regions. J Neurosci. 2006;26:2434–2442. doi: 10.1523/JNEUROSCI.4080-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gracia-Llanes FJ, Crespo C, Blasco-Ibanez JM, Marques-Mari AI, Martinez-Guijarro FJ. VIP-containing deep short-axon cells of the olfactory bulb innervate interneurons different from granule cells. Eur J Neurosci. 2003;18:1751–1763. doi: 10.1046/j.1460-9568.2003.02895.x. [DOI] [PubMed] [Google Scholar]
  25. Hanstein R, Lu A, Wurst W, Holsboer F, Deussing JM, Clement AB, Behl C. Transgenic overexpression of corticotropin releasing hormone provides partial protection against neurodegeneration in an in vivo model of acute excitotoxic stress. Neuroscience. 2008;156:712–721. doi: 10.1016/j.neuroscience.2008.07.034. [DOI] [PubMed] [Google Scholar]
  26. Hensch TK, Fagiolini M, Mataga N, Stryker MP, Baekkeskov S, Kash SF. Local GABA circuit control of experience-dependent plasticity in developing visual cortex. Science. 1998;282:1504–1508. doi: 10.1126/science.282.5393.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hitoshi S, Maruta N, Higashi M, Kumar A, Kato N, Ikenada K. Antidepressant drugs reverse the loss of adult neural stem cells following chronic stress. J. Neurosci Res. 2007;85:3574–3585. doi: 10.1002/jnr.21455. [DOI] [PubMed] [Google Scholar]
  28. Huang L, Garcia I, Jen HI, Arenkiel BR. Reciprocal connectivity between mitral cells and external plexiform layer interneurons in the mouse olfactory bulb. Front Neural Circuits. 2013;7:32. doi: 10.3389/fncir.2013.00032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Isaacson JS, Strowbridge BW. Olfactory reciprocal synapses: dendritic signaling in the CNS. Neuron. 1998;20:749–768. doi: 10.1016/s0896-6273(00)81013-2. [DOI] [PubMed] [Google Scholar]
  30. Jacobson L, Muglia LJ, Weninger SC, Pacak K, Majzoub JA. CRH deficiency impairs but does not block pituitary-adrenal responses to diverse stressors. Neuroendocrinology. 2000;71:79–87. doi: 10.1159/000054524. [DOI] [PubMed] [Google Scholar]
  31. Justice NJ, Yuan ZF, Sawchenko PE, Vale W. Type 1 corticotropin-releasing factor receptor expression reported in BAC transgenic mice: implications for reconciling ligand-receptor mismatch in the central corticotropin-releasing factor system. J Comp Neurol. 2008;511:479–496. doi: 10.1002/cne.21848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kato HK, Gillet SN, Peters AJ, Isaacson JS, Komiyama T. Parvalbumin expressing interneurons linearly control olfactory bulb output. Neuron. 2013;80:1218–1231. doi: 10.1016/j.neuron.2013.08.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kelsch W, Lin CW, Mosley CP, Lois C. A critical period for activity-dependent synaptic development during olfactory bulb adult neurogenesis. J Neurosci. 2009;29:11852–11858. doi: 10.1523/JNEUROSCI.2406-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kosaka T, Kosaka K. Heterogeneity of parvalbumin-containing neurons in the mouse main olfactory bulb, with special reference to short-axon cells and betaIV-spectrin positive dendritic segments. Neurosci Res. 2008;60:56–72. doi: 10.1016/j.neures.2007.09.008. [DOI] [PubMed] [Google Scholar]
  35. Kuhne C, Puk O, Graw J, Hrabe de Angelis M, Schutz G, Wurst W, Deussing JM. Visualizing corticotropin-releasing hormone receptor type 1 expression and neuronal connectivities in the mouse using a novel multifunctional allele. J Comp Neurol. 2012;520(14):3150–80. doi: 10.1002/cne.23082. [DOI] [PubMed] [Google Scholar]
  36. Lazarini F, Mouthon MA, Gheusi G, de Chaumont F, Olivo-Marin JC, Lamarque S, Abrous DN, Boussin FD, Lledo PM. Cellular and behavioral effects of cranial irradiation of the subventricular zone in adult mice. PLoS One. 2009;4:e7017. doi: 10.1371/journal.pone.0007017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Le Magueresse C, Monyer H. GABAergic interneurons shape the functional maturation of the cortex. Neuron. 2013;77:388–405. doi: 10.1016/j.neuron.2013.01.011. [DOI] [PubMed] [Google Scholar]
  38. Lemasson M, Saghatelyan A, Olivo-Marin JC, Lledo PM. Neonatal and adult neurogenesis provide two distinct populations of newborn neurons to the mouse olfactory bulb. J. Neurosci. 2005;25:6816–6825. doi: 10.1523/JNEUROSCI.1114-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Le Roux N, Cabezas C, Bohm UL, Poncer JC. Input-specific learning rules at excitatory synapses onto hippocampal parvalbumin-expressing interneurons. J Physiol. 2013;591:1809–1822. doi: 10.1113/jphysiol.2012.245852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lepousez G, Csaba Z, Bernard V, Loudes C, Videau C, Lacombe J, Epelbaum J, Viollet C. Somatostatin interneurons delineate the inner part of the external plexiform layer in the mouse main olfactory bulb. J Comp Neurol. 518:1976–1994. doi: 10.1002/cne.22317. [DOI] [PubMed] [Google Scholar]
  41. Lepousez G, Mouret A, Loudes C, Epelbaum J, Viollet C. Somatostatin contributes to in vivo gamma oscillation modulation and odor discrimination in the olfactory bulb. J Neurosci. 2010b;30:870–875. doi: 10.1523/JNEUROSCI.4958-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lipschitz DL, Crowley WR, Armstrong WE, Bealer SL. Neurochemical bases of plasticity in the magnocellular oxytocin system during gestation. Exp Neurol. 2005;196:210–223. doi: 10.1016/j.expneurol.2005.08.003. [DOI] [PubMed] [Google Scholar]
  43. Lledo PM, Merkle FT, Alvarez-Buylla A. Origin and function of olfactory bulb interneuron diversity. Trends Neurosci. 2008;31:392–400. doi: 10.1016/j.tins.2008.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ma Y, Hu H, Berrebi AS, Mathers PH, Agmon A. Distinct subtypes of somatostatin-containing neocortical interneurons revealed in transgenic mice. J Neurosci. 2006;26:5069–5082. doi: 10.1523/JNEUROSCI.0661-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, Hsu YW, Garcia AJ, 3rd, Gu X, Zanella S, et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci. 2012;15:793–802. doi: 10.1038/nn.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Maras PM, Baram TZ. Sculpting the hippocampus from within: stress, spines, and CRH. Trends Neurosci. 2012;35:315–324. doi: 10.1016/j.tins.2012.01.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Miyamichi K, Shlomai-Fuchs Y, Shu M, Weissbourd BC, Luo L, Mizrahi A. Dissecting local circuits: parvalbumin interneurons underlie broad feedback control of olfactory bulb output. Neuron. 2013;80(5):1232–1245. doi: 10.1016/j.neuron.2013.08.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ming GL, Song H. Adult neurogenesis in the mammalian central nervous system. Annu rev Neurosci. 2005;28:223–250. doi: 10.1146/annurev.neuro.28.051804.101459. [DOI] [PubMed] [Google Scholar]
  49. Mori K, Kishi K, Ojima H. Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb. J Comp Neurol. 1983;219:339–355. doi: 10.1002/cne.902190308. [DOI] [PubMed] [Google Scholar]
  50. Mouret A, Gheusi G, Gabellec MM, de Chaumont F, Olivo-Marin JC, Lledo PM. Learning and survival of newly generated neurons: when time matters. J Neurosci. 2008;28:11511–11516. doi: 10.1523/JNEUROSCI.2954-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Mouret A, Lepousez G, Gras J, Gabellec MM, Lledo PM. Turnover of newborn olfactory bulb neurons optimizes olfaction. J Neurosci. 2009;29:12302–12314. doi: 10.1523/JNEUROSCI.3383-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Muglia L, Jacobson L, Dikkes P, Majzoub JA. Corticotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature. 1995;373:427–432. doi: 10.1038/373427a0. [DOI] [PubMed] [Google Scholar]
  53. Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A. 2003;100:13940–13945. doi: 10.1073/pnas.1936192100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Nielsen SM, Nielsen LZ, Hjorth SA, Perrin MH, Vale WW. Constitutive activation of tethered-peptide/corticotropin-releasing factor receptor chimeras. Proc Natl Acad Sci U S A. 2000;97:10277–10281. doi: 10.1073/pnas.97.18.10277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Panzanelli P, Bardy C, Nissant A, Pallotto M, Sassoe-Pognetto M, Lledo PM, Fritschy JM. Early synapse formation in developing interneurons of the adult olfactory bulb. J Neurosci. 2009;29:15039–15052. doi: 10.1523/JNEUROSCI.3034-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Perrin MH, Donaldson CJ, Chen R, Lewis KA, Vale WW. Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor. Endocrinology. 1993;133:3058–3061. doi: 10.1210/endo.133.6.8243338. [DOI] [PubMed] [Google Scholar]
  57. Perrin M, Donaldson C, Chen R, Blount A, Berggren T, Bilezikjian L, Sawchenko P, Vale W. Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA. 1995;92:2969–2973. doi: 10.1073/pnas.92.7.2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Perrin MH, Vale WW. Corticotropin releasing factor receptors and their ligand family. Ann N Y Acad Sci. 1999;885:312–328. doi: 10.1111/j.1749-6632.1999.tb08687.x. [DOI] [PubMed] [Google Scholar]
  59. Pressler RT, Strowbridge BW. Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb. Neuron. 2006;49:889–904. doi: 10.1016/j.neuron.2006.02.019. [DOI] [PubMed] [Google Scholar]
  60. Price JL, Powell TP. An electron-microscopic study of the termination of the afferent fibres to the olfactory bulb from the cerebral hemisphere. J Cell Sci. 1970a;7:157–187. doi: 10.1242/jcs.7.1.157. [DOI] [PubMed] [Google Scholar]
  61. Price JL, Powell TP. The synaptology of the granule cells of the olfactory bulb. J Cell Sci. 1970b;7:125–155. doi: 10.1242/jcs.7.1.125. [DOI] [PubMed] [Google Scholar]
  62. Refojo D, Schweizer M, Kuehne C, Ehrenberg S, Thoeringer C, Vogl AM, Dedic N, et al. Glutamatergic and dopaminergic neurons mediate anxiogenic and anxiolytic effects of CRHR1. Science. 2011;333:1903–1907. doi: 10.1126/science.1202107. [DOI] [PubMed] [Google Scholar]
  63. Rochefort C, Gheusi G, Vincent JD, Lledo PM. Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory. J Neurosci. 2002;22:2679–2689. doi: 10.1523/JNEUROSCI.22-07-02679.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Roozendaal B, Schelling G, McGaugh JL. Corticotropin-releasing factor in the basolateral amygdala enhances memory consolidation via an interaction with the beta-adrenoceptor-cAMP pathway: dependence on glucocorticoid receptor activation. J Neurosci. 2008;28:6642–6651. doi: 10.1523/JNEUROSCI.1336-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Rudy B, Fishell G, Lee S, Hjerling-Leffler J. Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons. Dev Neurobiol. 2011;71:45–61. doi: 10.1002/dneu.20853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Schmolesky MT, De Ruiter MM, De Zeeuw CI, Hansel C. The neuropeptide corticotropin-releasing factor regulates excitatory transmission and plasticity at the climbing fibre-Purkinje cell synapse. Eur J Neurosci. 2007;25:1460–1466. doi: 10.1111/j.1460-9568.2007.05409.x. [DOI] [PubMed] [Google Scholar]
  67. Schoenfeld TJ, Gould E. Differential effects of stress and glucocorticoids on adult neurogenesis. Curr Top Behav Neurosci. 2013;15:139–164. doi: 10.1007/7854_2012_233. [DOI] [PubMed] [Google Scholar]
  68. Sheng H, Xu Y, Chen Y, Zhang Y, Xu X, He C, Ni X. CRH-R1 and CRH-R2 differentially modulate dendritic outgrowth of hippocampal neurons. Endocrine. 2012;41:458–464. doi: 10.1007/s12020-012-9603-5. [DOI] [PubMed] [Google Scholar]
  69. Shepherd GM, Greer CA. In: Olfactory bulb in the synaptic organization of the brain. Shepherd GM, editor. Oxford UP; New York: 2004. pp. 159–204. [Google Scholar]
  70. Smith GW, Aubry JM, Dellu F, Contarino A, Bilezikjian LM, Gold LH, Chen R, Marchuk Y, Hauser C, Bentley CA, et al. Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron. 1998;20:1093–1102. doi: 10.1016/s0896-6273(00)80491-2. [DOI] [PubMed] [Google Scholar]
  71. Takatoh J, Nelson A, Zhou X, Bolton MM, Ehlers MD, Arenkiel BR, Mooney R, Wang F. New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking. Neuron. 2013;77:346–360. doi: 10.1016/j.neuron.2012.11.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K, Kvitsiani D, Fu Y, Lu J, Lin Y, et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron. 2011;71:995–1013. doi: 10.1016/j.neuron.2011.07.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Thiel G, Cibelli G. Corticotropin-releasing factor and vasoactive intestinal polypeptide activate gene transcription through the cAMP signaling pathway in a catecholaminergic immortalized neuron. Neurochem Int. 1999;34:183–191. doi: 10.1016/s0197-0186(98)00086-2. [DOI] [PubMed] [Google Scholar]
  74. Tobin VA, Hashimoto H, Wacker DW, Takayanagi Y, Langnaese K, Caquineau C, Noack J, Landgraf R, Onaka T, Leng G, et al. An intrinsic vasopressin system in the olfactory bulb is involved in social recognition. Nature. 2010;464:413–417. doi: 10.1038/nature08826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB. Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature. 1998;391:892–896. doi: 10.1038/36103. [DOI] [PubMed] [Google Scholar]
  76. Turrigiano GG, Nelson SB. Homeostatic plasticity in the developin nervous system. Nat Rev Neurosci. 2004;5:97–107. doi: 10.1038/nrn1327. [DOI] [PubMed] [Google Scholar]
  77. Ung K, Arenkiel BR. Fiber-optic implantation for chronic optogenetic stimulation of brain tissue. J Vis Exp. 2012:e50004. doi: 10.3791/50004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Vale W, Rivier C, Brown MR, Spiess J, Koob G, Swanson L, Bilezikjian L, Bloom F, Rivier J. Chemical and biological characterization of corticotropin releasing factor. Recent Prog Horm Res. 1983;39:245–270. doi: 10.1016/b978-0-12-571139-5.50010-0. [DOI] [PubMed] [Google Scholar]
  79. Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science. 1981;213:1394–1397. doi: 10.1126/science.6267699. [DOI] [PubMed] [Google Scholar]
  80. Whitman MC, Greer CA. Synaptic integration of adult-generated olfactory bulb granule cells: basal axodendritic centrifugal input precedes apical dendrodendritic local circuits. J Neurosci. 2007;27:9951–9961. doi: 10.1523/JNEUROSCI.1633-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Wickersham IR, Finke S, Conzelmann KK, Callaway EM. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods. 2007a;4:47–49. doi: 10.1038/NMETH999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wickersham IR, Lyon DC, Barnard RJ, Mori T, Finke S, Conzelmann KK, Young JA, Callaway EM. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron. 2007b;53:639–647. doi: 10.1016/j.neuron.2007.01.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Xu H, Jeong HY, Tremblay R, Rudy B. Neocortical somatostatin-expressing GABAergic interneurons disinhibit the thalamorecipient layer 4. Neuron. 2013;77:155–167. doi: 10.1016/j.neuron.2012.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Yamaguchi M, Mori K. Critical period for sensory experience-dependent survival of newly generated granule cells in the adult mouse olfactory bulb. Proc Natl Acad Sci USA. 2005;102:9697–9702. doi: 10.1073/pnas.0406082102. [DOI] [PMC free article] [PubMed] [Google Scholar]

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